Tumour necrosis factor-alpha is a homotrimeric pro-inflammatory cytokine involved in systemic inflammation which exists in both soluble and membrane-bound forms. TNF-alpha is secreted predominantly by monocytes and macrophages but is also secreted by tumour cell lines as well as CD4+ and CD8+ peripheral blood T lymphocytes and some cultured T and B cell lines. TNF-alpha has been implicated in inflammatory diseases, autoimmune diseases, viral, bacterial and parasitic infections, malignancies, and/or neurodegenerative diseases and is a target for specific biological therapy in autoimmune/autoinflammatory diseases such as rheumatoid arthritis and Crohn's disease.
Crohn's disease, also known as Crohn syndrome and regional enteritis, is a type of inflammatory bowel disease causing a wide variety of symptoms. It primarily causes abdominal pain, diarrhea, vomiting and/or weight loss but may also cause complications outside the gastrointestinal tract (GIT) such as anaemia, skin rashes, arthritis, inflammation of the eye, tiredness, and lack of concentration (Baumgart et al 2012 The Lancet 380(9853):1590-605, herein incorporated by reference in its entirety). Crohn's disease is a presently incurable life-long gastrointestinal disease that is difficult to control with conventional therapies. Crohn's disease is discussed in more detail below under ‘autoimmune diseases’.
A TNF-alpha inhibitor which has sufficient specificity to TNF-alpha may be an efficient prophylactic or therapeutic pharmaceutical for preventing or treating diseases such as Crohn's disease, where TNF-alpha has been implicated as a key cytokine driving the pathology observed.
Antibody-based therapeutics have significant potential as effective treatments for autoimmune disease because they have high specificity for their target and a low inherent toxicity. Methods of treating autoimmune disease by administration of an antibody which binds TNF-alpha have been described (Kamm et al 2011 Inflamm Bowel Dis 17:2366-91, herein incorporated by reference in its entirety).
Three anti-TNF-alpha antibodies infliximab (trade name Remicade), adalimumab (trade name Humira) and certolizumab (or ‘tertolizumab pegol’, both trade name Cimzia) are used clinically for the treatment of Crohn's disease; however these antibodies are generally considered to be unsuitable for administration as oral therapeutics due to their inherent instability and susceptibility to proteolytic degradation by the digestive system, inflammatory proteases present at the sites of pathology in the intestinal tract, and intestinal microflora. These agents therefore have to be administered by intravenous infusion or subcutaneous injection which requires specialist training in order to use a hypodermic syringe or needle correctly and safely. These agents also require sterile equipment, a liquid formulation of the therapeutic polypeptide, vial packing of said polypeptide in a sterile and stable form and a suitable site on the subject for entry of the needle. Subjects commonly experience psychological stress before receiving an injection and pain while receiving an injection. Long term treatment with these systemic anti-TNF-alpha antibodies carries increased risks of serious infection and cancer. Together with the high costs of production, these factors currently restrict use of these agents to patients with more severe disease.
Several small molecule anti-inflammatory and immunosuppressive drugs are also currently in clinical development for Crohn's disease (Danese 2012 Gut 61:918-932 and Shealy et al 2010 mAbs 2:428-439, herein incorporated by reference in its entirety). Although these drugs are orally administered, many will be absorbed systemically after administration and may therefore have systemic immunosuppressive actions that are unrelated to actions against the gastrointestinal tract lesions. Furthermore, as small molecules lack the specificity of antibodies the risk of significant off target side-effects remains high.
Crohn's disease is primarily a disease of the gastrointestinal tract. The production of TNF-alpha is localised to cells present within mucosal and sub-mucosal tissues and this drives chronic inflammatory processes within the gut wall and the recruitment of additional inflammatory cells that are responsible for development of the disease immunopathology (van Deventer 1999 Ann Rheum Dis 58(Suppl I):I114-I120). The ability to deliver an oral therapeutic agent with high selectivity for TNF-alpha, but with exposure and activity limited to the gut, may offer efficacy similar to injectable anti-TNF-alpha antibodies, combined with significant improvements in safety due to reduced systemic exposure.
WO 2004/041862, WO 2006/122786 and Coppieters et al 2006 Arthritis & Rheumatism 54(6):1856-1866 (herein incorporated by reference in their entirety) disclose single domain antibodies directed against TNF-alpha and related aspects. The sequence referred to in WO 2006/122786 as “TNF1”, “PMP1 C2” or “SEQ ID NO: 52”) is characterised further below.
Polypeptides of the present invention may, in at least some embodiments, have one or more of the following advantages compared to anti-TNF-alpha substances of the prior art:                (i) increased affinity for TNF-alpha;        (ii) increased specificity for TNF-alpha;        (iii) increased neutralising capability against TNF-alpha;        (iv) increased cross-reactivity with TNF-alpha from different species such as human and cynomolgus monkey;        (v) increased cross-reactivity with both soluble and membrane forms of TNF-alpha;        (vi) reduced immunogenicity, for example when administered to a mouse, cynomolgus monkey or human;        (vii) increased stability in the presence of proteases, for example (a) in the presence of proteases found in the small and/or large intestine and/or IBD inflammatory proteases, for example trypsin, chymotrypsin, MMP3, MMP10, MMP12, other MMPs and cathepsin and/or (b) in the presence of proteases from gut commensal microflora and/or pathogenic bacteria, actively secreted and/or released by lysis of microbial cells found in the small and/or large intestine;        (viii) increased stability to protease degradation during production (for example resistance to yeast proteases)        (ix) increased suitability for oral administration;        (x) increased suitability for local delivery to the intestinal tract and lamina propria following oral administration;        (xi) increased suitability for expression, in a heterologous host such as bacteria such as Escherichia coli, or a yeast belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris;         (xii) suitability for, and improved properties for, use in a pharmaceutical;        (xiii) suitability for, and improved properties for, use in a functional food;        (xiv) improved tissue penetration such as penetration of inflamed colonic mucosal epithelium and submucosal tissues to access the sub mucosal lamina propria;        (xv) remain substantially active after (a) freezing and thawing and/or (b) after long term storage in lyophilised, liquid/cream format at for example 37 or 50 degrees C.;        (xvi) decreased immunogenicity in humans for example due to increased sequence similarity to human immunoglobulins;        (xvii) increased suitability for formatting in a multispecific format;        (xviii) binding to novel epitopes.        
Advantages (i) to (xviii) above may potentially be realised by the polypeptides of the present invention in a monovalent format or in a multivalent format such as a bihead format (for example homobihead or heterobihead formats).